Preclinical development of complex processing systems for ex vivo culture-expanded lymphohematopoietic cells, with subsequent immunologic and/or genetic manipulation, have been carried out in collaboration with a number of NIH Institute investigators. Preparation of CD8- depleted, culture-expanded lymphocytes: In collaboration with NIAID and NCHGR, this 10-day process that uses OKT3 + IL2 for T cell culture/expansion has been applied to a clinical trial of HIV gene therapy in the syngeneic twin model. The first phase of the clinical trial was completed in June 1998. In the past year, several methodologic innovations have been evaluated, including evaluation of new (non-Neo) vectors, T cell culture/expansion with CD3/CD28 beads (June method-see below) to enhance T cell function and longevity, and use of fibronectin-coated bags for improved gene transduction (see below). Preparation of allogeneic donor lymphocytes selectively depleted for alloreactive T cells: This process is being developed, in collaboration with NHLBI, for application to clinical allogeneic hematopoietic transplantation, especially in the HLA-mismatched setting. We had previously developed a reliable method (using OKT3 + IL2) for expan-sion of selected T cells from patients with chronic myelologenous leukemia, resulting in a product that has minimal contamination with leukemia cells and is capable of stimu-lating a third-party (donor) immune response to recipient-specific antigens. To date, five clinical scale expansions have been done, and the expansion characteristics appear to vary with the severity of the patients leukemia. 1999 Annual Report of Research Activities T lymphocyte culture/expansion in Aastrom Bioreactor: Over the past year, studies were initiated with the goal of adapting the T cell culture process described in (B) above to the Aastrom Replicell bioreactor system. To date, three culture-expansion procedures have been done using a manual version of the Replicell system; this developmental work will continue into FY2000. Novel methods for culture/expansion of lymphocytes: Over the past year, we have initiated studies to adapt the T cell culture/expansion method of Carl June (University of Pennsyvania) to NIH clinical protocol needs. This method uses an immunomagnetic bead coated with both OKT3 and CD28 to select and expand the T cell population with the goal of eliminating anergy or apoptosis that have been shown to occur with the tradi-tional (OKT3 + IL2) expansion method. Another immunomagnetic method that combines depletion of CD8 (T-suppressor) and CD20 (B) cells prior to culture is being developed for application to the HIV syngeneic twin studies and NCI studies of post-transplantation immunotherapy. Fibronectin transduction: A method for improved gene transduction using fibronectin-coated bags was developed and incorporated into the clinical trial of gene therapy for chronic granulomatous disease and will soon be used in several other clinical trials.